表没食子儿茶素没食子酸酯对大鼠脊髓损伤急性期的保护作用及机制
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摘要
目的：建立第8胸椎(T8)半切型脊髓损伤(spinal cord injury, SCI)大鼠模型,并采用过氧化氢(hydrogen peroxide, H2O2)诱导氧化应激损伤的PC12细胞作为神经损伤的细胞模型,从抗氧化、抗炎、抗凋亡等方面探讨表没食子儿茶素没食子酸酯(Epigallocatechin-3-gallate, EGCG)对脊髓损伤急性期的保护作用及其可能机制。
方法：从在体和离体两方面来研究EGCG对脊髓损伤的影响。
1在体实验：
(1)半切型脊髓损伤模型的建立及分组与给药
成年雌性SD大鼠T8处半切脊髓,造成SCI模型后随机分为6组：假手术组(暴露脊髓,但不进行半切)、模型组(SCI组)、阳性药组(甲基强的松龙,methyllpredriisolone sodiu succinate, MPSS,100mg/kg)、EGCG低剂量组(25mg／kg)、EGCG中剂量组(50 mg／kg)、EGCG高剂量组(100 mg／kg)。动物SCI术后5 min,假手术组和模型组给予生理盐水(10 ml／kg,ip),其余各组ip给予生理盐水配制的药物。
(2)SCI急性期脊髓组织病理组织学及超微结构的观察
SCI大鼠给药24 h后以损伤处为中心取下2 cm脊髓,放入含1 ml多聚甲醛溶液的EP管中避光保存做HE染色的病理切片；以损伤处为中心取下1 cm脊髓,用面刀切成1 mm3的小块置于2.5% 0.5 ml的戊二醛溶液中做透射电镜。
(3)SCI急性期血清炎症因子的检测
SCI大鼠给药24 h后摘右眼球取血5 ml,取血清,采用ELISA法测定血清白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)、细胞间粘附分子-1(ICAM-1)含量。
(4)SCI急性期抗氧化酶系统与活性氧簇的测定
SCI大鼠给药24 h后取脊髓制备成1%的组织匀浆,根据试剂盒说明书检测脊髓中SOD、MDA、O.2-、NO水平。取脊髓组织制备成凝胶电泳样本,采用Western blot法测定脊髓中iNOS蛋白表达水平。
(5)SCI急性期凋亡有关因子B-细胞淋巴瘤／白血病-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)的检测
SCI大鼠给药24 h后,取脊髓组织制备成凝胶电泳样本,采用Western blot法测定脊髓中Bcl-2和Bax蛋白表达。
(6)SCI急性期内源性神经营养因子营养因子-3(NT-3)和脑源性神经营养因子(BDNF)的测定
SCI大鼠给药24 h后,取脊髓组织制备成凝胶电泳样本,采用Western blot法测定脊髓中内源性神经营养因子NT-3和BDNF蛋白表达。
2离体实验：
(1)H2O2诱导的细胞损伤模型的建立
PC12细胞以2×105／cm2接种在50 ml细胞培养瓶,常规培养,培养液每天更换一次,48h内当细胞汇集至80%时进行传代接种。细胞加药处理前采用清饥饿法使之同步化。加入H2O2(终浓度100～600μmol／L)孵育2 h,用噻唑蓝(MTT)法确立PC 12细胞损伤模型。
(2)EGCG对H2O2诱导氧化应激损伤的PC12细胞的保护作用
细胞用EGCG和H2O2处理后,收集细胞培养液,根据乳酸脱氢酶(LDH)试剂盒说明检测细胞外LDH水平,用以检测细胞膜通透性及完整性；采用MTT法观察细胞线粒体完整性与细胞活力；收集EGCG和H2O2处理后的细胞,用流式细胞术测定细胞周期分布以及细胞增殖率；将细胞接种至内有爬片的6孔培养板,孵育、同步化,用EGCG和H2O2处理后,吸弃上清液,每孔加入10μmol／L BrdU液1 ml,孵育24 h,BrdU免疫荧光法检测细胞周期相S期DNA增生。
(3)EGCG对H2O2诱导氧化应激损伤的PC12细胞保护作用的机制
收集EGCG和H2O2处理后细胞,试剂盒测定细胞内抗氧化酶系统超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)的水平。同时根据试剂盒说明书依次测定活性氧簇(reactive oxygen species, ROS)中超氧自由基(O.2-)、羟自由基(·OH)、诱导型一氧化氮合成酶(iNOS)的活性与一氧化氮(NO)含量。采用Western blot检测iNOS蛋白水平,采用real timeRT-PCR测定iNOS-mRNA水平。
结果：从离体和在体两方面结果来研究EGCG对脊髓损伤的影响。
1在体实验：
(1)EGCG对脊髓损伤急性期脊髓组织病理组织学及超微结构的影响
HE染色后光学显微镜下观察发现,假手术组大鼠脊髓灰、白质组织结构完整,SCI组脊髓组织切片在损伤区中央灰质可见大片血细胞,并有大量单核或多核的炎性细胞浸润,并可出现囊腔样变化。EGCG低、中剂量组脊髓组织空泡变性减轻,可见血细胞与炎症细胞,以单核或多核炎性细胞为主,但明显低于SCI组。EGCG高剂量组与阳性对照药MPSS组,损伤部位脊髓组织结构排列较完整,偶见炎性水肿,损伤脊髓组织未见明显细胞凋亡,白质内神经纤维排列整齐。
透射电子显微镜观察了脊髓组织神经元和轴突。假手术组脊髓灰质内神经元与白质各索髓鞘规则。SCI组脊髓灰质内神经元胞体固缩,白质髓鞘排列松散。EGCG 25～50 mg／kg组脊髓灰质内能频繁见到胞体肿胀的神经元,髓鞘变薄排列松散。EGCG 100 mg／kg组脊髓灰质内神经元与白质髓鞘结构基本正常。
(2)EGCG对脊髓损伤急性期血清炎症因子的影响
大鼠脊髓半切24 h后,SCI组血清炎症因子IL-1β、IL-6、IL-8、ICAM-1、TNF-α含量增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,血清IL-1β、IL-6、ICAM-1、TNF-α含量均减少,与SCI组比较有统计学差异(P<0.05),血清IL-8含量亦减少,与SCI组比较,EGCG 25 mg／kg无统计学差异(P>0.05),50、100 mg／kg有显著统计学差异(P<0.01)。
(3)EGCG抗氧化作用
大鼠脊髓半切24 h后,SCI组脊髓组织SOD水平下降,MDA水平增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织SOD水平增加,MDA水平减少,与SCI组比较有统计学差异(P<0.05)。
大鼠脊髓半切24 h后,SCI组脊髓组织抑制O.2-活性能力下降,NO水平显著增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织抑制O.2-活性能力增加,与SCI组比较有统计学差异(P<0.05);EGCG三个浓度亦减少脊髓组织NO水平,与SCI组比较,EGCG 25 mg／kg剂量组无统计学差异(P>0.05),EGCG 50、100 mg／kg剂量组有显著统计学差异(P<0.01)。Western blot实验结果显示,大鼠脊髓半切24 h后,SCI组脊髓组织iNOS蛋白表达水平增加,与假手术组比较有显著统计学差异(P<0.01),EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织iNOS蛋白表达水平减少,与SCI组比较有显著统计学差异(P<0.01)。
(4)EGCG抗凋亡作用
Western blot实验结果显示,大鼠脊髓半切24 h后,SCI组脊髓组织Bcl-2蛋白表达水平下降,Bax蛋白表达水平与Bax/Bcl-2蛋白表达比率增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织Bcl-2蛋白表达水平增加,Bax蛋白表达水平与Bax/Bcl-2蛋白表达比率减少,其中,与模型组比较,25 mg／kg组没有统计学差异(P>0.05),EGCG 50、100 mg／kg剂量组有统计学差异(P<0.05)。
(5)EGCG对脊髓损伤后急性期神经营养因子NT-3和BDNF的影响
Western blot实验结果显示,大鼠脊髓半切24 h后,脊髓组织NT-3、BDNF蛋白表达水平下降,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织NT-3、BDNF蛋白表达水平增加,其中,与模型组比较,25 mg／kg组没有统计学差异(P>0.05),EGCG 50、100 mg／kg剂量组有统计学差异(P<0.05)。
2离体实验：
(1)H2O2诱导的PC12细胞损伤模型
倒置显微镜下观,空白对照组细胞贴壁生长牢固,体积饱满,充分伸展,细胞明亮清晰,折光性好,而损伤组PC12细胞体积明显缩小,突起消失,胞体变小,细胞间隙增大,细胞分布变稀疏的,折光性下降,细胞皱缩,出现不同程度的脱壁,出现明显损伤形态。损伤程度以600μmol／L H2O2最严重的。MTT实验结果显示,与空白对照组相比,100～300μmol／L H2O2剂量能降低细胞活力,但是没有统计学意义,400～600μmol／L剂量能降低细胞活力,且具有显著统计学意义(P<0.01)。
(2)EGCG对H2O2诱导氧化应激损伤的PC12细胞的保护作用
LDH水平反映了细胞膜完整性的程度,膜外LDH水平高,则细胞膜受损程度高。500μmol／L H2O2孵育PC12细胞2 h,膜外LDH水平升高,与空白对照组比较有显著统计学差异(P<0.01)。EGCG预孵1 h,5、10、20μmol／L剂量组能降低膜外LDH水平,与H2O2组比较有统计学差异(P<0.05)。
MTT法测定受损PC12细胞活力与线粒体的完整性,结果显示,EGCG 5-30μmol／L浓度组能显著改善500μmol／L H2O2所致的PC12细胞线粒体的损伤,能显著增加受损细胞的活力,与H2O2组比较有统计学差异(P<0.05)。
流式细胞术结果显示,500μmol／L H2O2孵育PC12细胞2 h,G0／G1期细胞分布百分率增加,细胞周期S期细胞分布百分率与细胞增殖率减少,与空白对照组比较有显著统计学差异(P<0.01)。EGCG预孵1 h,5、10、20μmol／L三个剂量能降低G0／G1期细胞分布百分率,升高细胞增殖率,与H2O2组比较有统计学差异(P<0.05)。EGCG三个剂量组能升高S期细胞分布百分率,与H2O2组比较,5μmol／L组无统计学差异(P>0.05),10μmol／L与20μmol／L组有显著统计学差异(P<0.01)。
S期DNA合成通过BrdU免疫荧光染色实验来检测。500μmol／L H2O2孵育PC12细胞2 h,BrdU阳性细胞数减少,与空白对照组比较有显著统计学差异(P<0.01)。EGCG预处理1 h,5、10、20μmol／L三个剂量组能升高BrdU阳性细胞数,与H2O2组比较有显著统计学差异(P<0.01)。
(3)EGCG对H2O2诱导损伤PC12细胞保护作用的机制
细胞经H2O2诱导损伤后,SOD、GSH-Px水平下降,MDA水平上升,与空白对照组比较有统计学差异(P<0.05)。EGCG预孵1h,5、10、20μmol／L三个剂量组细胞内SOD、GSH-Px水平上升,MDA水平下降,与H2O2组比较有显著统计学差异(P<0.01)。
细胞经H2O2诱导损伤后,细胞内抑制O.2-活性能力下降,产生·OH能力增加,与空白对照组比较有显著统计学差异(P<0.01),EGCG预孵1 h,5、10、20μmol／L三个剂量组细胞内抑制O.2-活性能力增加,产生OH能力减少,与H2O2组比较有显著统计学差异(P<0.01)；细胞经H2O2诱导损伤后,细胞外NO水平与细胞内iNOS活性上升,与空白对照组比较有显著统计学差异(P<0.01),EGCG预孵1 h,三个剂量组细胞内iNOS活性与细胞外NO水平下降,与H2O2组比较有显著统计学差异(P<0.01)；Western blot与real time RT-PCR实验结果显示,细胞经H2O2诱导损伤后,细胞细胞内iNOS蛋白与mRNA表达上升,与空白对照组比较有显著统计学差异(P<0.01),EGCG三个剂量组细胞内iNOS蛋白与mRNA表达水平下降,与H2O2组比较,5μmol／L无统计学差异(P>0.05),10μmol／L与20μmol／L两剂量组有统计学差异(P<0.05)。说明EGCG能有效拮抗H2O2导致的iNOS蛋白与mRNA表达的增加,能抑制iNOS-NO通路的激活。
结论：我们采用T8处半切大鼠脊髓造成SCI体内损伤模型,以及采用H2O2诱导PC12细胞损伤造成神经损伤体外模型,在体与离体给予EGCG干预,通过生化指标及病理的指标观察EGCG对脊髓损伤的作用,主要结论如下：
(1)EGCG能保护SCI大鼠受损脊髓,这种保护作用源于：减少SCI大鼠血清炎症因子含量,有显著抗炎作用；提高SCI大鼠脊髓中抗氧化酶系统活性,抑制氧自由基及iNOS-NO通路的激活而发挥抗氧化作用；下调凋亡基因Bax的表达,上调抗凋亡基因Bcl-2的表达而产生抗凋亡作用；同时促进内源性神经营养因子NT-3、BDNF的表达。
(2)EGCG能够维持受损PC12细胞膜完整性,保护细胞的线粒体,延长细胞周期的S期及促进该期DNA合成,增加受损细胞的增殖率。这种神经保护作用源于EGCG提高SOD、GSH-Px等抗氧化酶活性,降低ROS活性,抑制iNOS-NO通路的激活。
Objective:To explore the effects and its possible mechanisms of Epigallocatechin-3-gallate (EGCG) on the acute phase in spinal cord injury (SCI) in rats, several aspects of the anti-oxidative, anti-inflammatory, anti-apoptotic effect were studied in the present study by using hemisected SCI rats at thoracic vertebra 8 (T8) as SCI rat model and hydrogen peroxide (H2O2)-induced PC 12 cells with oxidative-stress injury as the neuronal damaga cell model.
Methods:The present study investigated the effects of EGCG on SCI model from in vivo and in vitro.
1 Experiment in vivo:
(1) Spinal cord injury procedure, grouping and treating
Spinal cord was hemisected at thoracic vertebra 8 (T8) in adult female Sprague-Dawley rats. Rats were randomly divided into six different groups as follows:(1) sham-treated rats (saline,5 ml/kg, ip), (2) SCI (saline,5 ml/kg, ip), (3) Methyllprednisolone sodium succinate (MPSS,100 mg/kg, ip), (4) EGCG (25 mg/kg, ip), (5) EGCG (50 mg/kg, ip) and (6) EGCG (100 mg/kg, ip) with a single dose immediately following trauma. MPSS and EGCG were diluted in saline.
(2) Micro-morphological examination by pathohistology observation by HE staining and transmission electron microscopic in SCI rats
Twenty-four hours after SCI and administration on rats, the spinal cord with 2 cm was fixed in 4% buffered paraformaldehyde to observe pathohistology change. The spinal cord with 1 cm was obtained by abscising vertebral canal, and 1 mm3 cross-section of gray and white matter at the epicenter of each spinal cord lesion was fixed in 2.5% glutaraldehyde to observe micro-morphology.
(3) Determination the concentration of serum inflammatory factor by ELISA assay in SCI rats Twenty-four hours after SCI and administration on rats, animals were=removed right eyeball and exanguinated 5 ml. The serum was collected. ELISA assay was performed to determine the concentration of serum Interleukin-1β(IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor necrosis factor-α(TNF-α) and Intercellular adhesion molecule-1 (ICAM-1) using commercial kit according to the description of the manufacturer.
(4) Measurement of the levels of SOD, MDA, O.2-, NO and iNOS protein in SCI rats
Twenty-four hours after SCI and administration on rats, spinal cord was made 1% tissue homogenate to detect the levels of SOD, MDA, O.2- and NO according to the manufacturer's instruction. The spinal cords from each rat were made into gel electrophoresis samples to determine iNOS protein levels by Western blot assay.
(5) Measusement of B-cell lymphoma/leukemia-2 gene B (Bcl-2) and Bcl 2-associated X protein (Bax) protein levels by Western blot in SCI rats
Twenty-four hours after SCI and administration on rats, the spinal cords from each rat were made into gel electrophoresis samples to determine Bcl-2 and Bax protein levels by Western blot assay.
(6) Measusement of NT-3 and BDNF protein levels by Western blot in SCI rats
Twenty-four hours after SCI and administration on rats, the spinal cords from each rat were made into gel electrophoresis samples to determine NT-3 and BDNF protein levels by Western blot assay.
2 Experiment in vitro:
(1) PC12 cells culture and injury procedure
PC 12 cells were plated at a density of 2×105 cells/cm2 in a 50 ml culture flask and cultured routinely. The culture media were changed every 24 h. The cells were subcultured when attached upon reaching 80～90% confluence in 48 h. The cells were synchronized by serum starvation before EGCG and H2O2 treatment. Varying concentrations of H2O2 (final concentration 100～600μmol/L) were added to each well and co-incubated for 2 h. Damaged PC 12 cells model was established by MTT assay.
(2) The protective effects of EGCG on damaged PC 12 cells
Being treated with EGCG and H2O2, the culture media were collected and tested depending on LDH kits to measure of extracellular LDH levels. The cellular viability and mitochondria integrity were detected by MTT assay. Being treated with EGCG and H2O2, the cell cycle was monitor by cytometry. The cells were seeded onto poly-L-lysinated 18×18 cm rectangular coverslips placed in 6-well plates (2×105 cells/ml in 1ml medium), and cultured, synchronized and treated with EGCG and H2O2. The media were replaced with serum-free DMEM media containing BrdU at a final concentration of 10μmol/L for 24 h. DNA synthesis of S phase in cell cycle was determinated by BrdU-labeled immunofluorescence assay.
(3) The mechanism of EGCG on oxidative-stress damaged PC 12 cells
Being treated with EGCG and H2O2, PC 12 cells were collected. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) enzymes, hydroxy radical (·OH) and superoxide anion (O.2-), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in PC12 cells were measured according to the kits. iNOS protein and mRNA levels were determinated by Western blot and real-time RT-PCR.
Results:The present study investigated the effects of EGCG on SCI model from in vivo and in vitro.
1 Experiment in vivo:
(1) The effect of EGCG on micro-morphology and pathohistology in acute SCI rats
The neuronal and axonal pathohistology with HE staining was observed by the optical microscope. In sham group, the construction in grey matter and white matter was normal size and regular. In SCI group, some blood cells appeared in central gray of damaged area. A lot of monokaryotic or polycaryotic inflammatory cells infiltrated into grey matter and white matter, with capsular space change. In EGCG 25～50 mg/kg group, vacuolar degeneration was lightened, along with blood cells and inflammatory cells, and lower than the one in SCI group. In EGCG 100 mg/kg group and positive drug MPSS, myeloid tissue in damaged part arranged in regular way, inflammatory edema and apoptosis appeared occasionally.The nerve fiber in white matter arranged in regular way.
The neuronal and axonal changes were observed by transmission electron microscopy. In sham group, the spinal cord motoneurons of the anterior grey horn and myeline sheaths in the white matter were normal size and regular. In SCI group, the spinal cord motoneurons of the grey horn shrinked back, and myeline sheaths in the white matter arranged in loose. In EGCG 25～50 mg/kg group, the spinal cord motoneurons of the anterior grey horn appeared frequently oncotic. Myeline sheaths in the white matter arranged in loose way, along with primary demyelination. In EGCG 100 mg/kg group and positive drug MPSS, motoneurons and axon were almost normal size and regular.
(2) Effect of EGCG on the concentration of serum inflammatory factor in acute SCI rats
Twenty-four hours after SCI, the concentration of serum inflammatory factor IL-1β, IL-6, IL-8, TNF-αand ICAM-1 increased significantly in SCI model group (P < 0.01 vs sham group), while the concentrations were reduced by 25,50 and 100 mg/kg EGCG treatment except the treatment effect of 25 mg/kg EGCG on serum IL-8 (P< 0.05 vs SCI group).
(3) Antioxidation of EGCG in acute SCI rats
Antioxidase system was measured according to the kits. The results showed that SOD levels was reduced and MDA levels increased in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG increased SOD levels, reduced MDA levels (P < 0.05 vs SCI group).
Reactive oxygen species were measured according to the kits. The capability of suppressing O.2- activity was lowed severely, and NO levels were expressed highly in SCI model group (P< 0.01 vs sham group). The capability of suppressing O.2-activity was increased by 25,50 and 100 mg/kg EGCG treatment (P< 0.05 vs SCI group). NO levels were reduced by three dosages of EGCG treatment as compared with SCI model group, with no statistical significance in 25 100 mg/kg (P> 0:05 vs SCI group), and the statistical significance in 50 and 100 mg/kg (P< 0.01 vs SCI group). Western blot assays showed that the levels of iNOS protein were expressed excessively in SCI model group (P< 0.01 vs sham group), along with significantly decreased by EGCG treatment (P< 0.01 vs SCI group).
(4) Anti-apoptosis effect of EGCG in acute SCI rats
Western blot assays showed that B-cell lymphoma/leukemia-2 (Bcl-2) levels were depressed, and Bax levels and the ratio of Bax to Bcl-2 expression were expressed excessively in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG reversed the changes, along with 25 mg/kg EGCG no statistical significance (P>0.05 vs SCI group),50 mg/kg and 100 mg/kg EGCG statistical significance (P< 0.05 vs SCI group).
(5) Effect of EGCG on the neurotrophic factor neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) in acute SCI rats
Western blot assays showed that NT-3 and BDNF levels were depressed excessively in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG increased the expression levels, along with 25 mg/kg EGCG no statistical significance,50 mg/kg and 100 mg/kg EGCG statistical significance (P< 0.05 vs SCI group).
2 Experiment in vitro:
(1) H2O2-induced PC 12 cells damaged model
Observed by inverted microscope, the Blank group cells with full size attached solidly to the wall of culture flask, and stretched full. Cells body with good refractive index was bright and clear. The damaged cells body shrinked back, accompaniment with enation disappearance, cell spaces augmentation and sparsate distribution. The obviously damaged morphous were showed in the damaged cells. The degree of injury was the severest in the 600μmol/L H2O2 group. MTT asays showed that H2O2 decreased PC 12 cells viability as compared with the control group, with the statistical significance in 400～600μmol/L (P<0.01 vs Control group), and no statistical significance in 100～300μmol/L (P> 0.05 vs Control group).
(2) The neuroprotective effect of EGCG on damaged PC 12 cells
An increase in the amount of membrane damaged cells resulted in an increase of LDH enzyme activity and membrane permeability. Extracellular LDH levels were enhanced by H2O2 treatment (P< 0.01 vs Control group). Three dosages of EGCG decreased membrane permeability by depressing the LDH activity (P< 0.05 vs H2O2 group).
The cellular viability and mitochondria integrity were detected by MTT assay, and the results showed that 5～30μmol/L EGCG could antagonize the mitochondrial injury and the decreasing viability induced by H2O2 except the 2.5 umol/L EGCG group, significantly higher than the H2O2 group (P< 0.05 vs H2O2 group).
Flow cytometry assay showed that the percentage of G0/G1-phase cells was increased, and the percentage of S-phase cells and Proliferative Index decreased by H2O2 treatment (P< 0.01 vs Control group). Three dosages of EGCG decreased the percentage of G0/G1-phase cells, increased Proliferative Index (P< 0.05 vs H2O2 group). Three dosages of EGCG also increased the percentage of S-phase cells as compared with Control group, with the statistical significance in 10,20μmol/L (P<0.01 vs H2O2 group), and no statistical significance in 5μmol/L (P> 0.05 vs H2O2 group).
To detect further proliferating cells in the S-phase, BrdU incorporated in the cells was measured. The number of BrdU-positive cells decreased significantly in cells treated with H2O2 (P< 0.01 vs Control group), while the number of BrdU-positive cells increased in cells with EGCG pretreatment (P< 0.01 vs H2O2 group).
(3)The neuroprotective mechanism of EGCG on H2O2-induced damaged PC 12 cells
Antioxidase system was measured according to the kits. The results showed that SOD and GSH-Px levels were reduced and MDA levels increased in H2O2-induced PC 12 cells (P< 0.05 vs Control group). The levels of SOD and GSH-Px were increased and the contents of MDA reduced by EGCG pretreatment (P< 0.01 vs H2O2 group).
Reactive oxygen species (ROS) were measured according to the kits. The results showed that the capability of suppressing O.2- activity was lowed, and the capability of producing·OH was enhanced in H2O2 group (P< 0.01 vs Control group). Three dosages of EGCG reversed the changes (P< 0.01 vs H2O2 group). Extracellular NO contents and intracellular iNOS activity were expressed highly in H2O2 group (P< 0.01 vs Control group), and reduced by EGCG pretreatment in damaged cells (P< 0.01 vs H2O2 group). Western blot andreal time RT-PCR assays showed that the levels of protein and mRNA of iNOS were expressed excessively in H2O2 group (P< 0.01 vs Control group), along with significantly decreased by EGCG pretreatment except the 5μmol/L group(P< 0.05 vs H2O2 group).
Conclusion:In the present study we studied the effects of EGCG on acute SCI model from the aspects of biochemical and pathological indicator by using hemisected SCI rats as SCI model in vivo and H2O2-induced PC 12 cells oxidative-stress injury as the neuronal damaga model in vitro. The conclusion as follows:
(1) EGCG played an important role in spinal cord injury at the early stages. The neuroprotective effect attributed to several aspects:anti-inflammatory effect by decreasing the concentration of serum inflammatory factor in acute SCI rats; antioxidation by enhancing antioxidase system activity in spinal cord, inhibiting ROS activity, and depressing the activation of iNOS-NO pathway; anti-apoptosis effect by downregulating Bax expression and upregulating Bcl-2 expression; promoting endogenous neurotrophic factor NT-3 and BDNF expression.
(2) The present study demonstrated that EGCG was able to protect H2O2-induced PC 12 cells by enhancing antioxidase SOD, GSH-Px activity, decreasing ROS activity, and depressing the activation of iNOS-NO pathway, which could decrease membrane permeability, maintain membrane integrity, protect mitochondria, prolong the S phase in cell cycle and promote DNA synthesis of S phase.
方法：从在体和离体两方面来研究EGCG对脊髓损伤的影响。
1在体实验：
(1)半切型脊髓损伤模型的建立及分组与给药
成年雌性SD大鼠T8处半切脊髓,造成SCI模型后随机分为6组：假手术组(暴露脊髓,但不进行半切)、模型组(SCI组)、阳性药组(甲基强的松龙,methyllpredriisolone sodiu succinate, MPSS,100mg/kg)、EGCG低剂量组(25mg／kg)、EGCG中剂量组(50 mg／kg)、EGCG高剂量组(100 mg／kg)。动物SCI术后5 min,假手术组和模型组给予生理盐水(10 ml／kg,ip),其余各组ip给予生理盐水配制的药物。
(2)SCI急性期脊髓组织病理组织学及超微结构的观察
SCI大鼠给药24 h后以损伤处为中心取下2 cm脊髓,放入含1 ml多聚甲醛溶液的EP管中避光保存做HE染色的病理切片；以损伤处为中心取下1 cm脊髓,用面刀切成1 mm3的小块置于2.5% 0.5 ml的戊二醛溶液中做透射电镜。
(3)SCI急性期血清炎症因子的检测
SCI大鼠给药24 h后摘右眼球取血5 ml,取血清,采用ELISA法测定血清白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)、细胞间粘附分子-1(ICAM-1)含量。
(4)SCI急性期抗氧化酶系统与活性氧簇的测定
SCI大鼠给药24 h后取脊髓制备成1%的组织匀浆,根据试剂盒说明书检测脊髓中SOD、MDA、O.2-、NO水平。取脊髓组织制备成凝胶电泳样本,采用Western blot法测定脊髓中iNOS蛋白表达水平。
(5)SCI急性期凋亡有关因子B-细胞淋巴瘤／白血病-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)的检测
SCI大鼠给药24 h后,取脊髓组织制备成凝胶电泳样本,采用Western blot法测定脊髓中Bcl-2和Bax蛋白表达。
(6)SCI急性期内源性神经营养因子营养因子-3(NT-3)和脑源性神经营养因子(BDNF)的测定
SCI大鼠给药24 h后,取脊髓组织制备成凝胶电泳样本,采用Western blot法测定脊髓中内源性神经营养因子NT-3和BDNF蛋白表达。
2离体实验：
(1)H2O2诱导的细胞损伤模型的建立
PC12细胞以2×105／cm2接种在50 ml细胞培养瓶,常规培养,培养液每天更换一次,48h内当细胞汇集至80%时进行传代接种。细胞加药处理前采用清饥饿法使之同步化。加入H2O2(终浓度100～600μmol／L)孵育2 h,用噻唑蓝(MTT)法确立PC 12细胞损伤模型。
(2)EGCG对H2O2诱导氧化应激损伤的PC12细胞的保护作用
细胞用EGCG和H2O2处理后,收集细胞培养液,根据乳酸脱氢酶(LDH)试剂盒说明检测细胞外LDH水平,用以检测细胞膜通透性及完整性；采用MTT法观察细胞线粒体完整性与细胞活力；收集EGCG和H2O2处理后的细胞,用流式细胞术测定细胞周期分布以及细胞增殖率；将细胞接种至内有爬片的6孔培养板,孵育、同步化,用EGCG和H2O2处理后,吸弃上清液,每孔加入10μmol／L BrdU液1 ml,孵育24 h,BrdU免疫荧光法检测细胞周期相S期DNA增生。
(3)EGCG对H2O2诱导氧化应激损伤的PC12细胞保护作用的机制
收集EGCG和H2O2处理后细胞,试剂盒测定细胞内抗氧化酶系统超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)的水平。同时根据试剂盒说明书依次测定活性氧簇(reactive oxygen species, ROS)中超氧自由基(O.2-)、羟自由基(·OH)、诱导型一氧化氮合成酶(iNOS)的活性与一氧化氮(NO)含量。采用Western blot检测iNOS蛋白水平,采用real timeRT-PCR测定iNOS-mRNA水平。
结果：从离体和在体两方面结果来研究EGCG对脊髓损伤的影响。
1在体实验：
(1)EGCG对脊髓损伤急性期脊髓组织病理组织学及超微结构的影响
HE染色后光学显微镜下观察发现,假手术组大鼠脊髓灰、白质组织结构完整,SCI组脊髓组织切片在损伤区中央灰质可见大片血细胞,并有大量单核或多核的炎性细胞浸润,并可出现囊腔样变化。EGCG低、中剂量组脊髓组织空泡变性减轻,可见血细胞与炎症细胞,以单核或多核炎性细胞为主,但明显低于SCI组。EGCG高剂量组与阳性对照药MPSS组,损伤部位脊髓组织结构排列较完整,偶见炎性水肿,损伤脊髓组织未见明显细胞凋亡,白质内神经纤维排列整齐。
透射电子显微镜观察了脊髓组织神经元和轴突。假手术组脊髓灰质内神经元与白质各索髓鞘规则。SCI组脊髓灰质内神经元胞体固缩,白质髓鞘排列松散。EGCG 25～50 mg／kg组脊髓灰质内能频繁见到胞体肿胀的神经元,髓鞘变薄排列松散。EGCG 100 mg／kg组脊髓灰质内神经元与白质髓鞘结构基本正常。
(2)EGCG对脊髓损伤急性期血清炎症因子的影响
大鼠脊髓半切24 h后,SCI组血清炎症因子IL-1β、IL-6、IL-8、ICAM-1、TNF-α含量增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,血清IL-1β、IL-6、ICAM-1、TNF-α含量均减少,与SCI组比较有统计学差异(P<0.05),血清IL-8含量亦减少,与SCI组比较,EGCG 25 mg／kg无统计学差异(P>0.05),50、100 mg／kg有显著统计学差异(P<0.01)。
(3)EGCG抗氧化作用
大鼠脊髓半切24 h后,SCI组脊髓组织SOD水平下降,MDA水平增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织SOD水平增加,MDA水平减少,与SCI组比较有统计学差异(P<0.05)。
大鼠脊髓半切24 h后,SCI组脊髓组织抑制O.2-活性能力下降,NO水平显著增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织抑制O.2-活性能力增加,与SCI组比较有统计学差异(P<0.05);EGCG三个浓度亦减少脊髓组织NO水平,与SCI组比较,EGCG 25 mg／kg剂量组无统计学差异(P>0.05),EGCG 50、100 mg／kg剂量组有显著统计学差异(P<0.01)。Western blot实验结果显示,大鼠脊髓半切24 h后,SCI组脊髓组织iNOS蛋白表达水平增加,与假手术组比较有显著统计学差异(P<0.01),EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织iNOS蛋白表达水平减少,与SCI组比较有显著统计学差异(P<0.01)。
(4)EGCG抗凋亡作用
Western blot实验结果显示,大鼠脊髓半切24 h后,SCI组脊髓组织Bcl-2蛋白表达水平下降,Bax蛋白表达水平与Bax/Bcl-2蛋白表达比率增加,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织Bcl-2蛋白表达水平增加,Bax蛋白表达水平与Bax/Bcl-2蛋白表达比率减少,其中,与模型组比较,25 mg／kg组没有统计学差异(P>0.05),EGCG 50、100 mg／kg剂量组有统计学差异(P<0.05)。
(5)EGCG对脊髓损伤后急性期神经营养因子NT-3和BDNF的影响
Western blot实验结果显示,大鼠脊髓半切24 h后,脊髓组织NT-3、BDNF蛋白表达水平下降,与假手术组比较有显著统计学差异(P<0.01)。EGCG 25、50、100 mg／kg三个浓度分别作用于SCI大鼠24 h,脊髓组织NT-3、BDNF蛋白表达水平增加,其中,与模型组比较,25 mg／kg组没有统计学差异(P>0.05),EGCG 50、100 mg／kg剂量组有统计学差异(P<0.05)。
2离体实验：
(1)H2O2诱导的PC12细胞损伤模型
倒置显微镜下观,空白对照组细胞贴壁生长牢固,体积饱满,充分伸展,细胞明亮清晰,折光性好,而损伤组PC12细胞体积明显缩小,突起消失,胞体变小,细胞间隙增大,细胞分布变稀疏的,折光性下降,细胞皱缩,出现不同程度的脱壁,出现明显损伤形态。损伤程度以600μmol／L H2O2最严重的。MTT实验结果显示,与空白对照组相比,100～300μmol／L H2O2剂量能降低细胞活力,但是没有统计学意义,400～600μmol／L剂量能降低细胞活力,且具有显著统计学意义(P<0.01)。
(2)EGCG对H2O2诱导氧化应激损伤的PC12细胞的保护作用
LDH水平反映了细胞膜完整性的程度,膜外LDH水平高,则细胞膜受损程度高。500μmol／L H2O2孵育PC12细胞2 h,膜外LDH水平升高,与空白对照组比较有显著统计学差异(P<0.01)。EGCG预孵1 h,5、10、20μmol／L剂量组能降低膜外LDH水平,与H2O2组比较有统计学差异(P<0.05)。
MTT法测定受损PC12细胞活力与线粒体的完整性,结果显示,EGCG 5-30μmol／L浓度组能显著改善500μmol／L H2O2所致的PC12细胞线粒体的损伤,能显著增加受损细胞的活力,与H2O2组比较有统计学差异(P<0.05)。
流式细胞术结果显示,500μmol／L H2O2孵育PC12细胞2 h,G0／G1期细胞分布百分率增加,细胞周期S期细胞分布百分率与细胞增殖率减少,与空白对照组比较有显著统计学差异(P<0.01)。EGCG预孵1 h,5、10、20μmol／L三个剂量能降低G0／G1期细胞分布百分率,升高细胞增殖率,与H2O2组比较有统计学差异(P<0.05)。EGCG三个剂量组能升高S期细胞分布百分率,与H2O2组比较,5μmol／L组无统计学差异(P>0.05),10μmol／L与20μmol／L组有显著统计学差异(P<0.01)。
S期DNA合成通过BrdU免疫荧光染色实验来检测。500μmol／L H2O2孵育PC12细胞2 h,BrdU阳性细胞数减少,与空白对照组比较有显著统计学差异(P<0.01)。EGCG预处理1 h,5、10、20μmol／L三个剂量组能升高BrdU阳性细胞数,与H2O2组比较有显著统计学差异(P<0.01)。
(3)EGCG对H2O2诱导损伤PC12细胞保护作用的机制
细胞经H2O2诱导损伤后,SOD、GSH-Px水平下降,MDA水平上升,与空白对照组比较有统计学差异(P<0.05)。EGCG预孵1h,5、10、20μmol／L三个剂量组细胞内SOD、GSH-Px水平上升,MDA水平下降,与H2O2组比较有显著统计学差异(P<0.01)。
细胞经H2O2诱导损伤后,细胞内抑制O.2-活性能力下降,产生·OH能力增加,与空白对照组比较有显著统计学差异(P<0.01),EGCG预孵1 h,5、10、20μmol／L三个剂量组细胞内抑制O.2-活性能力增加,产生OH能力减少,与H2O2组比较有显著统计学差异(P<0.01)；细胞经H2O2诱导损伤后,细胞外NO水平与细胞内iNOS活性上升,与空白对照组比较有显著统计学差异(P<0.01),EGCG预孵1 h,三个剂量组细胞内iNOS活性与细胞外NO水平下降,与H2O2组比较有显著统计学差异(P<0.01)；Western blot与real time RT-PCR实验结果显示,细胞经H2O2诱导损伤后,细胞细胞内iNOS蛋白与mRNA表达上升,与空白对照组比较有显著统计学差异(P<0.01),EGCG三个剂量组细胞内iNOS蛋白与mRNA表达水平下降,与H2O2组比较,5μmol／L无统计学差异(P>0.05),10μmol／L与20μmol／L两剂量组有统计学差异(P<0.05)。说明EGCG能有效拮抗H2O2导致的iNOS蛋白与mRNA表达的增加,能抑制iNOS-NO通路的激活。
结论：我们采用T8处半切大鼠脊髓造成SCI体内损伤模型,以及采用H2O2诱导PC12细胞损伤造成神经损伤体外模型,在体与离体给予EGCG干预,通过生化指标及病理的指标观察EGCG对脊髓损伤的作用,主要结论如下：
(1)EGCG能保护SCI大鼠受损脊髓,这种保护作用源于：减少SCI大鼠血清炎症因子含量,有显著抗炎作用；提高SCI大鼠脊髓中抗氧化酶系统活性,抑制氧自由基及iNOS-NO通路的激活而发挥抗氧化作用；下调凋亡基因Bax的表达,上调抗凋亡基因Bcl-2的表达而产生抗凋亡作用；同时促进内源性神经营养因子NT-3、BDNF的表达。
(2)EGCG能够维持受损PC12细胞膜完整性,保护细胞的线粒体,延长细胞周期的S期及促进该期DNA合成,增加受损细胞的增殖率。这种神经保护作用源于EGCG提高SOD、GSH-Px等抗氧化酶活性,降低ROS活性,抑制iNOS-NO通路的激活。
Objective:To explore the effects and its possible mechanisms of Epigallocatechin-3-gallate (EGCG) on the acute phase in spinal cord injury (SCI) in rats, several aspects of the anti-oxidative, anti-inflammatory, anti-apoptotic effect were studied in the present study by using hemisected SCI rats at thoracic vertebra 8 (T8) as SCI rat model and hydrogen peroxide (H2O2)-induced PC 12 cells with oxidative-stress injury as the neuronal damaga cell model.
Methods:The present study investigated the effects of EGCG on SCI model from in vivo and in vitro.
1 Experiment in vivo:
(1) Spinal cord injury procedure, grouping and treating
Spinal cord was hemisected at thoracic vertebra 8 (T8) in adult female Sprague-Dawley rats. Rats were randomly divided into six different groups as follows:(1) sham-treated rats (saline,5 ml/kg, ip), (2) SCI (saline,5 ml/kg, ip), (3) Methyllprednisolone sodium succinate (MPSS,100 mg/kg, ip), (4) EGCG (25 mg/kg, ip), (5) EGCG (50 mg/kg, ip) and (6) EGCG (100 mg/kg, ip) with a single dose immediately following trauma. MPSS and EGCG were diluted in saline.
(2) Micro-morphological examination by pathohistology observation by HE staining and transmission electron microscopic in SCI rats
Twenty-four hours after SCI and administration on rats, the spinal cord with 2 cm was fixed in 4% buffered paraformaldehyde to observe pathohistology change. The spinal cord with 1 cm was obtained by abscising vertebral canal, and 1 mm3 cross-section of gray and white matter at the epicenter of each spinal cord lesion was fixed in 2.5% glutaraldehyde to observe micro-morphology.
(3) Determination the concentration of serum inflammatory factor by ELISA assay in SCI rats Twenty-four hours after SCI and administration on rats, animals were=removed right eyeball and exanguinated 5 ml. The serum was collected. ELISA assay was performed to determine the concentration of serum Interleukin-1β(IL-1β), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor necrosis factor-α(TNF-α) and Intercellular adhesion molecule-1 (ICAM-1) using commercial kit according to the description of the manufacturer.
(4) Measurement of the levels of SOD, MDA, O.2-, NO and iNOS protein in SCI rats
Twenty-four hours after SCI and administration on rats, spinal cord was made 1% tissue homogenate to detect the levels of SOD, MDA, O.2- and NO according to the manufacturer's instruction. The spinal cords from each rat were made into gel electrophoresis samples to determine iNOS protein levels by Western blot assay.
(5) Measusement of B-cell lymphoma/leukemia-2 gene B (Bcl-2) and Bcl 2-associated X protein (Bax) protein levels by Western blot in SCI rats
Twenty-four hours after SCI and administration on rats, the spinal cords from each rat were made into gel electrophoresis samples to determine Bcl-2 and Bax protein levels by Western blot assay.
(6) Measusement of NT-3 and BDNF protein levels by Western blot in SCI rats
Twenty-four hours after SCI and administration on rats, the spinal cords from each rat were made into gel electrophoresis samples to determine NT-3 and BDNF protein levels by Western blot assay.
2 Experiment in vitro:
(1) PC12 cells culture and injury procedure
PC 12 cells were plated at a density of 2×105 cells/cm2 in a 50 ml culture flask and cultured routinely. The culture media were changed every 24 h. The cells were subcultured when attached upon reaching 80～90% confluence in 48 h. The cells were synchronized by serum starvation before EGCG and H2O2 treatment. Varying concentrations of H2O2 (final concentration 100～600μmol/L) were added to each well and co-incubated for 2 h. Damaged PC 12 cells model was established by MTT assay.
(2) The protective effects of EGCG on damaged PC 12 cells
Being treated with EGCG and H2O2, the culture media were collected and tested depending on LDH kits to measure of extracellular LDH levels. The cellular viability and mitochondria integrity were detected by MTT assay. Being treated with EGCG and H2O2, the cell cycle was monitor by cytometry. The cells were seeded onto poly-L-lysinated 18×18 cm rectangular coverslips placed in 6-well plates (2×105 cells/ml in 1ml medium), and cultured, synchronized and treated with EGCG and H2O2. The media were replaced with serum-free DMEM media containing BrdU at a final concentration of 10μmol/L for 24 h. DNA synthesis of S phase in cell cycle was determinated by BrdU-labeled immunofluorescence assay.
(3) The mechanism of EGCG on oxidative-stress damaged PC 12 cells
Being treated with EGCG and H2O2, PC 12 cells were collected. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) enzymes, hydroxy radical (·OH) and superoxide anion (O.2-), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in PC12 cells were measured according to the kits. iNOS protein and mRNA levels were determinated by Western blot and real-time RT-PCR.
Results:The present study investigated the effects of EGCG on SCI model from in vivo and in vitro.
1 Experiment in vivo:
(1) The effect of EGCG on micro-morphology and pathohistology in acute SCI rats
The neuronal and axonal pathohistology with HE staining was observed by the optical microscope. In sham group, the construction in grey matter and white matter was normal size and regular. In SCI group, some blood cells appeared in central gray of damaged area. A lot of monokaryotic or polycaryotic inflammatory cells infiltrated into grey matter and white matter, with capsular space change. In EGCG 25～50 mg/kg group, vacuolar degeneration was lightened, along with blood cells and inflammatory cells, and lower than the one in SCI group. In EGCG 100 mg/kg group and positive drug MPSS, myeloid tissue in damaged part arranged in regular way, inflammatory edema and apoptosis appeared occasionally.The nerve fiber in white matter arranged in regular way.
The neuronal and axonal changes were observed by transmission electron microscopy. In sham group, the spinal cord motoneurons of the anterior grey horn and myeline sheaths in the white matter were normal size and regular. In SCI group, the spinal cord motoneurons of the grey horn shrinked back, and myeline sheaths in the white matter arranged in loose. In EGCG 25～50 mg/kg group, the spinal cord motoneurons of the anterior grey horn appeared frequently oncotic. Myeline sheaths in the white matter arranged in loose way, along with primary demyelination. In EGCG 100 mg/kg group and positive drug MPSS, motoneurons and axon were almost normal size and regular.
(2) Effect of EGCG on the concentration of serum inflammatory factor in acute SCI rats
Twenty-four hours after SCI, the concentration of serum inflammatory factor IL-1β, IL-6, IL-8, TNF-αand ICAM-1 increased significantly in SCI model group (P < 0.01 vs sham group), while the concentrations were reduced by 25,50 and 100 mg/kg EGCG treatment except the treatment effect of 25 mg/kg EGCG on serum IL-8 (P< 0.05 vs SCI group).
(3) Antioxidation of EGCG in acute SCI rats
Antioxidase system was measured according to the kits. The results showed that SOD levels was reduced and MDA levels increased in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG increased SOD levels, reduced MDA levels (P < 0.05 vs SCI group).
Reactive oxygen species were measured according to the kits. The capability of suppressing O.2- activity was lowed severely, and NO levels were expressed highly in SCI model group (P< 0.01 vs sham group). The capability of suppressing O.2-activity was increased by 25,50 and 100 mg/kg EGCG treatment (P< 0.05 vs SCI group). NO levels were reduced by three dosages of EGCG treatment as compared with SCI model group, with no statistical significance in 25 100 mg/kg (P> 0:05 vs SCI group), and the statistical significance in 50 and 100 mg/kg (P< 0.01 vs SCI group). Western blot assays showed that the levels of iNOS protein were expressed excessively in SCI model group (P< 0.01 vs sham group), along with significantly decreased by EGCG treatment (P< 0.01 vs SCI group).
(4) Anti-apoptosis effect of EGCG in acute SCI rats
Western blot assays showed that B-cell lymphoma/leukemia-2 (Bcl-2) levels were depressed, and Bax levels and the ratio of Bax to Bcl-2 expression were expressed excessively in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG reversed the changes, along with 25 mg/kg EGCG no statistical significance (P>0.05 vs SCI group),50 mg/kg and 100 mg/kg EGCG statistical significance (P< 0.05 vs SCI group).
(5) Effect of EGCG on the neurotrophic factor neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) in acute SCI rats
Western blot assays showed that NT-3 and BDNF levels were depressed excessively in SCI model group (P< 0.01 vs sham group). Three dosages of EGCG increased the expression levels, along with 25 mg/kg EGCG no statistical significance,50 mg/kg and 100 mg/kg EGCG statistical significance (P< 0.05 vs SCI group).
2 Experiment in vitro:
(1) H2O2-induced PC 12 cells damaged model
Observed by inverted microscope, the Blank group cells with full size attached solidly to the wall of culture flask, and stretched full. Cells body with good refractive index was bright and clear. The damaged cells body shrinked back, accompaniment with enation disappearance, cell spaces augmentation and sparsate distribution. The obviously damaged morphous were showed in the damaged cells. The degree of injury was the severest in the 600μmol/L H2O2 group. MTT asays showed that H2O2 decreased PC 12 cells viability as compared with the control group, with the statistical significance in 400～600μmol/L (P<0.01 vs Control group), and no statistical significance in 100～300μmol/L (P> 0.05 vs Control group).
(2) The neuroprotective effect of EGCG on damaged PC 12 cells
An increase in the amount of membrane damaged cells resulted in an increase of LDH enzyme activity and membrane permeability. Extracellular LDH levels were enhanced by H2O2 treatment (P< 0.01 vs Control group). Three dosages of EGCG decreased membrane permeability by depressing the LDH activity (P< 0.05 vs H2O2 group).
The cellular viability and mitochondria integrity were detected by MTT assay, and the results showed that 5～30μmol/L EGCG could antagonize the mitochondrial injury and the decreasing viability induced by H2O2 except the 2.5 umol/L EGCG group, significantly higher than the H2O2 group (P< 0.05 vs H2O2 group).
Flow cytometry assay showed that the percentage of G0/G1-phase cells was increased, and the percentage of S-phase cells and Proliferative Index decreased by H2O2 treatment (P< 0.01 vs Control group). Three dosages of EGCG decreased the percentage of G0/G1-phase cells, increased Proliferative Index (P< 0.05 vs H2O2 group). Three dosages of EGCG also increased the percentage of S-phase cells as compared with Control group, with the statistical significance in 10,20μmol/L (P<0.01 vs H2O2 group), and no statistical significance in 5μmol/L (P> 0.05 vs H2O2 group).
To detect further proliferating cells in the S-phase, BrdU incorporated in the cells was measured. The number of BrdU-positive cells decreased significantly in cells treated with H2O2 (P< 0.01 vs Control group), while the number of BrdU-positive cells increased in cells with EGCG pretreatment (P< 0.01 vs H2O2 group).
(3)The neuroprotective mechanism of EGCG on H2O2-induced damaged PC 12 cells
Antioxidase system was measured according to the kits. The results showed that SOD and GSH-Px levels were reduced and MDA levels increased in H2O2-induced PC 12 cells (P< 0.05 vs Control group). The levels of SOD and GSH-Px were increased and the contents of MDA reduced by EGCG pretreatment (P< 0.01 vs H2O2 group).
Reactive oxygen species (ROS) were measured according to the kits. The results showed that the capability of suppressing O.2- activity was lowed, and the capability of producing·OH was enhanced in H2O2 group (P< 0.01 vs Control group). Three dosages of EGCG reversed the changes (P< 0.01 vs H2O2 group). Extracellular NO contents and intracellular iNOS activity were expressed highly in H2O2 group (P< 0.01 vs Control group), and reduced by EGCG pretreatment in damaged cells (P< 0.01 vs H2O2 group). Western blot andreal time RT-PCR assays showed that the levels of protein and mRNA of iNOS were expressed excessively in H2O2 group (P< 0.01 vs Control group), along with significantly decreased by EGCG pretreatment except the 5μmol/L group(P< 0.05 vs H2O2 group).
Conclusion:In the present study we studied the effects of EGCG on acute SCI model from the aspects of biochemical and pathological indicator by using hemisected SCI rats as SCI model in vivo and H2O2-induced PC 12 cells oxidative-stress injury as the neuronal damaga model in vitro. The conclusion as follows:
(1) EGCG played an important role in spinal cord injury at the early stages. The neuroprotective effect attributed to several aspects:anti-inflammatory effect by decreasing the concentration of serum inflammatory factor in acute SCI rats; antioxidation by enhancing antioxidase system activity in spinal cord, inhibiting ROS activity, and depressing the activation of iNOS-NO pathway; anti-apoptosis effect by downregulating Bax expression and upregulating Bcl-2 expression; promoting endogenous neurotrophic factor NT-3 and BDNF expression.
(2) The present study demonstrated that EGCG was able to protect H2O2-induced PC 12 cells by enhancing antioxidase SOD, GSH-Px activity, decreasing ROS activity, and depressing the activation of iNOS-NO pathway, which could decrease membrane permeability, maintain membrane integrity, protect mitochondria, prolong the S phase in cell cycle and promote DNA synthesis of S phase.
引文
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